A retrospective cohort study was performed on all patients ≥ 18 years of age who had received either PD-1 inhibitors (nivolumab or pembrolizumab) or CTLA-4 inhibitor (ipilimumab) with or without combination chemotherapy for advanced NSCLC at Upstate University Hospital, Syracuse, New York during the period from January 1, 2016 to December 31, 2020. Patient demographics, smoking history, clinical data, cancer diagnosis, and treatment history were collected. Data on antimicrobial use within four weeks prior to the initiation of anticancer therapy and the names of the antimicrobial used were also collected. Broad-spectrum antimicrobials were defined as antibiotics that are effective against both Gram-negative and Gram-positive organisms. We chose the 4-week time point to evaluate the effects of antibiotics on clinical benefit and survival, because any intestinal microbiome changes due to antibiotics would last longer than 4 weeks. The study was conducted with Institutional Review Board exemption from State University Upstate Medical University Institutional Review Board.

Our primary endpoint was clinical benefits and overall survival (OS). Clinical Benefit to therapy were determined by reviewing and comparing the imaging modality (CT or PET) at baseline and after starting therapy. Tumor responses were classified as Complete Response (CR) if there was a total resolution, Partial Response (PR) if at least 50% reduction, and Stable Disease (SD) if there was no significant change (<20% enlargement) or reduction of <50% of the tumor mass. No response to treatment was given if there was progression on imaging. Clinical Benefit was defined as CR + PR + SD. RECIST criteria were not used for response evaluation in our institution outside the context of clinical trials.

Overall survival (OS) measured the time from start of therapy to death due to any cause and progression-free survival (PFS) the time from start of therapy to radiologic and/or clinical disease progression necessitating change of therapy. Clinical Benefit rate was defined at the proportion of treated patients based on their best response during therapy.

The National Institute of Health (NIH) guidelines define MetS as having three or more of the following characteristics (19): 1. Large waist, at least 35 inches for women and 40 inches for men; 2. High triglyceride level of 150 mg/dL or 1.7 mmol/L, or higher; 3. Reduced level of high-density lipoprotein (HDL) cholesterol of less than 40 mg/dL or 1.04 mmol/L in men or less than 50 mg/dL or 1.3 mmol/L in women; 4. Hypertension, with blood pressure of 130/85 mm Hg or higher; and 5. Elevated fasting blood glucose of 100 mg/dL or 5.6 mmol/L or higher

Since waist circumference was not a clinical parameter readily available in the patient’s medical records, the following modified criteria was used for the diagnosis of MetS in this study: 1. Subjects with a diagnosis of MetS documented in the medical record was considered to have the disorder; and 2. In the absence of measurements for the waist circumference, it was assumed that patients with a body mass index (BMI) of 30 or higher would satisfy the NIH criteria for large waist given the high correlation between BMI and waist circumferences for either gender (22). BMIs in this study were calculated based on the weight and height of the patients just prior to starting lung cancer therapy.

Cox proportional hazards regression analysis were carried out to determine the hazard ratios (HRs) for the following covariates in PFS and OS: Prior use of broad-spectrum antimicrobials, age, tumor histology, metabolic syndrome, neutrophil:lymphocyte (N:L) ratio, obesity, PD-L1 expression, and smoking history. Results were presented individually in forest plots and as survival at mean of co-variates.

We next divided the 118 patients into two groups, based on whether they fulfilled the criteria for a diagnosis of MetS or not. We next determined whether the survival advantage associated with MetS occurred in both patients treated with ICI monotherapy and with chemoimmunotherapy. To do so, we stratified the patients according to whether they received first-line ICI monotherapy or chemoimmunotherapy for NSCLC. Finally, we determined if obesity was associated with improved outcome to ICI therapy in our cohort of patients.

The Clinical Benefit rate, progression-free survival (PFS) and OS were calculated. Survival was plotted as a time-dependent covariate using the Kaplan-Meier method. Association of factors potentially predictive of Clinical Benefit was evaluated using the Chi-square tests. Differences in the various clinical and laboratory parameters were calculated as mean and compared using the Student’s t tests. A two-sided p value of < 0.05 was considered statistically significant.

Between January 1, 2016 to December 31, 2020, a total of 712 patients were diagnosed with NSCLC at our institution. One hundred and eighty-four patients received first-line therapy with ICIs. However, only 118 patients were included for analysis because the other 66 patients did not have sufficient data in their medical record for the determination of their MetS status, treatment response or other clinical outcomes. To ensure that we did not select for those with different treatment outcomes, we compared the characteristics of the patients in these two groups. We did not find any significant difference in age, gender, ECOG score, tumor histologic type, therapeutic modality, PD-L1 expression, use of broad-spectrum antimicrobials, smoking history, and N:L ratio between the two groups of patients ( Table 1 ), supporting the notion that the cohort of patients included in the analysis was representative of the entire population of patients.

Ninety-one patients (77%) had adenocarcinoma, 20 (17%) squamous cell carcinoma, and 7 (6%) mixed adenosquamous carcinoma. There were 61 females and 57 males. Median age was 66 years (range 43-86), ECOG performance status 1 (range 0-3), and PD-L1 expression 50% (range 0-90%). Thirty-two (27%) patients received broad-spectrum antibiotics within four weeks of starting their first-line NSCLC therapy. Fifty-seven (48%) patients received ICI monotherapy and 61 (52%) chemoimmunotherapy. The therapy regimens used are shown in Table 2 . Despite being initiated on therapy, 50 patients (42%) did not respond to the treatment and they experienced progression of disease (PD).

We next determined the effects of the following a set of clinical parameters on the outcome of the entire group of analyzed patients: Prior broad-spectrum antimicrobial use, age, tumor histology, MetS, N:L ratio, obesity, PD-L1 expression, and smoking. None of the covariates, except PD-L1 expression, affected PFS of the patients in univariate analysis ( Table 3 ). However, MetS (p = 0.02), lower N:L ratio (p = 0.02), and higher PD-L1 expression (p = 0.05) were associated with lower HRs. Multivariate analysis showed PD-L1 expression (p = 0.011) to be the only factor ( Figure 1A ) that affected PFS favorably. In contrast, MetS (p = 0.022) and PD-L1 (p = 0.001) expression were associated with lower risks and prior use of broad-spectrum antimicrobials (p = 0.035) a higher risk for overall survival ( Figure 1B ). Table 4 shows the HRs for each of the covariates. These results suggest that, in addition to PD-L1 and prior use of broad-spectrum antimicrobials, MetS is an independent prognostic factor in patients with advanced lung cancer treated with immune checkpoint inhibitors +/- chemotherapy.

The results of the multivariate analysis led us to divide the 118 evaluable patients into two groups according to whether they had MetS or not to further evaluate the effects MetS had on treatment outcomes. Since the diagnosis of MetS requires at least three of the five criteria modified by us based on the NIH guidelines, only patients with a BMI > 30 and fulfilled at least two of the other four criteria, or those with BMI <30 but fulfilled at least three of the other four criteria were assigned the diagnosis of MetS. Based on these criteria, twenty-one (17.8%) patients were classified as having MetS and 97 (82.2%) non-MetS based on the available clinical characteristics. Table 5 shows the clinical characteristics of the two groups. They were comparable in gender distribution, age, ECOG performance status, histologic types of the tumor, therapeutic modality, prior use of broad-spectrum antimicrobials, or PD-L1 expression. With a median follow-up of 9 months (range 0.5-67), the group of patients with MetS enjoyed significantly longer OS (median = 22 months) compared to those who did not have MetS (median = 9 months) (HR 0.54, 95% CI: 0.31-0.92) (p = 0.02) ( Figure 2A ). As expected from the results of the multivariate analysis, there was no significant difference in the PFS between the two groups ( Figure 2B ), suggesting that patients with MetS who were treated with first-line ICIs might have responded or tolerated better to second-line therapy when their disease progressed than those who did not have MetS. The probabilities of clinical benefits/disease-control (Complete Response + Partial Response + Stable Disease) were comparable between the two groups of patients (71.4% vs 54.6%; p = 0.22).

We next determined whether the survival advantage associated with MetS occurred in both patients treated with ICI monotherapy and with chemoimmunotherapy. To do so, we stratified the patients according to whether they received first line ICI monotherapy or chemoimmunotherapy for NSCLC. Of the 57 patients who received only ICI monotherapy, 10 patients had MetS and 47 did not. There was no significant difference between the two groups in terms of gender distribution, age, ECOG performance status, histologic types of the tumor, or PD-L1 expression. However, 7/10 (70%) patients with MetS and10/47 (22.7%) of patients without MetS received broad-spectrum antimicrobials within four weeks of starting anti-NSCLC therapy (p = 0.005). Despite the high proportion of patients with MetS received broad-spectrum antimicrobials, patients with MetS still enjoyed significantly longer OS (median not reached) (HR 0.47, 95% CI: 0.22-1.00) (p = 0.05) ( Figure 3A ) but not PFS ( Figure 3B ) when compared to those without MetS (median = 8 months). In contrast, there was no significant difference in the OS ( Figure 4A ) or PFS ( Figure 4B ) in patients who were treated with first-line chemoimmunotherapy, whether the patients had MetS or not. The disease-control rates were also comparable between the two groups of patients irrespective of the treatment modality.

To dissect the effects of the prior use of broad-spectrum antimicrobials from those due to MetS in patients treated with ICI monotherapy, we performed a multivariate analysis of the two covariates for OS at two time points. MetS and not prior use of broad-spectrum antimicrobials predicted OS at six months (p = 0.043) and at twelve months (p = 0.008).

Twenty-nine (24.5%) patients had BMI ≥30 and fulfilled the criteria for obesity. Four patients who were classified as having MetS had BMIs <30, and 12 patients with a BMI >30 did not have MetS. There was no significant difference between the two groups of patients divided according to obesity in terms of gender distribution, age, ECOG performance status, histologic types of the tumor, treatment modality, or PD-L1 expression. Twelve of 29 patients with obesity (41.4%) and 19/89 (21%) patients without received broad-spectrum antimicrobials prior to starting anti-NSCLC therapy (p = 0.05). We did not find any significant difference in the OS or PFS between the two groups, nor did we find any difference in the probability of progression of disease despite being started on therapy in both groups. Finally, within the group of patients with MetS, we did not find any difference in the OS among patients who were obese and patients who were not obese (p = 0.22).